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Regular Articles |
From the Institute of Pathology,*
University of Berne, Berne,
Switzerland, and the Department of Integrative Biology and
Pharmacology,
University of Texas, Houston
Medical School, Houston, Texas
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Somatostatin receptors consist of a family of at least five different somatostatin receptor subtypes,7,8 which are currently being characterized functionally. These somatostatin receptor subtypes are present in normal somatostatin target tissues7 and are also found in various proportions in somatostatin-responsive human tumors.9-11 One of the subtypes frequently expressed by human tumors is sst2,10 as demonstrated by mRNA expression and ligand specificity. This observation is of clinical importance as sst2 is the human somatostatin receptor subtype with the highest affinity for commercially available, synthetic somatostatin analogues, such as octreotide.12 The 111In-labeled DTPA-octreotide radioligand is therefore particularly efficient in localizing in vivo sst2-expressing tumors,13,14 and octreotide therapy will be most efficient in sst2-expressing tumors.5,14
The in vitro identification of sst2 receptors in human pathological tissues, such as neoplasms, is therefore particularly important clinically. Up to now, two in vitro methods have been used to detect these receptors: 1) binding studies on tissue homogenates15 or tissue sections16 (receptor autoradiography) using sst2-preferring ligands such as 125I-labeled Tyr3-octreotide and 2) sst2 mRNA analysis using either in situ hybridization methods on tissue sections10,14 or reverse transcription polymerase chain reaction and RNAse protection assays on tissue homogenates.11,17,18 These two methodological approaches, however, require a considerable specialized expertise, are time-consuming, frequently involve radioactive material (125I or 32P), do not always provide a high cellular resolution, and can in only one case (in situ hybridization) be performed in formalin-fixed material. An alternative specific and sensitive method to identify sst2 receptors in formalin-fixed human tissue is presently not available and would obviously be of great clinical relevance.
Recently, Schonbrunn and colleagues have developed a polyclonal somatostatin receptor antibody that, when tested in sst-transfected cells and in rat brain and pancreatic tissues, was shown to be highly specific for sst2A receptors.19-21 The aim of the present study was therefore to evaluate this antibody immunohistochemically on tissue sections of human tumors, either formalin-fixed, frozen, or both, and to compare the results with those obtained using other available in vitro methods, namely, receptor autoradiography or in situ hybridization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two types of tumor samples were selected for this study. 1) Frozen
samples from 24 different tumors, which were characterized for their
somatostatin receptor content by receptor autoradiography using the
sst2-preferring 125I-labeled Tyr3-octreotide
and the universal somatostatin receptor ligand 125I-labeled
Leu8-DTrp22-Tyr25-somatostatin-28
(LTT-SS-28)16
(these tumors were also tested for their sst
mRNA using in situ hybridization10
whenever
possible). 2) Twenty-three other samples were divided into one piece
frozen immediately after resection and another piece fixed in formalin
for the routine histopathological diagnosis. The frozen piece of tissue
was used as described in 1) above. As shown in Tables 2 and 3
, these
tumors were divided into somatostatin receptor-positive and
somatostatin receptor-negative types; somatostatin receptor-positive
tumors were further divided into sst2-expressing and sst2-lacking
specimens, according to selective ligand binding and in situ
hybridization results.
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Frozen sections were incubated for 2 hours at room temperature with a 125I-labeled tyrosine-3 analogue of the somatostatin octapeptide octreotide or with the somatostatin-28 analogue, 125I-labeled LTT-SS-28, as described previously.16 After the sections were washed, they were apposed to 3H-Hyperfilms (Amersham, Little Chalfont, UK) and exposed for 1 week in x-ray cassettes.16 Nonspecific binding was determined in parallel sections incubated with the same concentration of labeled peptide in the presence of 10-6 mol/L of the corresponding unlabeled peptide. The autoradiograms were quantified using a computer-assisted image-processing system, as previously described.16
In Situ Hybridization
Cryostat sections (20 µm) were used for sst1, sst2, and sst3
mRNA detection by in situ hybridization. The protocol
followed was essentially that described in detail
previously,9,10
using the same oligonucleotide probes as
described earlier.9,10
They were labeled at the 3' end
using [
-32P]dATP (>3000 Ci/mmol; Amersham, Aylesbury,
UK) and terminal deoxynucleotidyltransferase (Boehringer Mannheim,
Mannheim, Germany) to specific activities of 0.9 x
104
to 2.0 x 104
Ci/mmol.10,22
All necessary controls were performed as
reported previously.10
Immunohistochemical Evaluation of the sst2A Antibody R2–88
The R2–88 rabbit polyclonal antibody was used as primary antibody. R2–88 was raised against a unique sequence in the carboxyl-terminal region of the sst2A receptor, corresponding to amino acids 339 to 359 in the rat protein.20 The identical sequence is found in the human, rat, and mouse sst2A receptor proteins, and as a result, the antibody is expected to recognize the receptor from all three species. Previous studies showed positive reactivity with the rat receptor.19-21 The antibody does not cross-react with any of the other sst receptor subtypes.19,20
Frozen Tissues
Ten-micron-thick sections, adjacent to the sections used for in vitro receptor autoradiography and in situ hybridization, were cut on a cryostat (Leitz).
The following basic protocol was used. The sections were fixed for 10 minutes in acetone, post-fixed for 10 minutes in 4% paraformaldehyde (diluted in PBS), and incubated for 20 minutes in 5% normal goat serum diluted in Tris-buffered saline (TBS). The sections were then incubated with the R2–88 antibody against the sst2A receptor overnight at room temperature. The antibody R2–88 was used at a 1:6000 dilution in TBS containing 1% bovine serum albumin, 5% normal goat serum, and 0.1% NaN3. Sections were then incubated in a 1:200 dilution (same buffer as for primary antibody) of biotinylated goat anti-rabbit immunoglobulin antiserum (Dako, Glostrup, Denmark) and thereafter with avidin-biotin complex/horseradish peroxidase (1:120 in TBS; Dako). Finally, sections were developed in 0.05% 3,3'-diaminobenzidine (Fluka, Buchs, Switzerland) and 0.006% H2O2 (Merck, Darmstadt, Germany), weakly counterstained with hematoxylin, and mounted. A tumor was considered to be positive for R2–88 when the immunostaining was abolished after absorption of the antibody with the peptide antigen at 100 nmol/L concentration (30 minutes at room temperature, with agitation before application of the antibody to the tissue). The tumor was considered negative if the immunostaining was not suppressed in the presence of the antigen. In preliminary experiments, titrations with different concentrations of antigen were performed, and antibody reactivity both on Western blots and in ELISAs was tested; 100 nmol/L peptide completely blocked the staining of the receptor protein on a Western blot, and in ELISAs, the peptide was bound with an EC50 of 5 nmol/L.20
As positive control for the immunohistochemistry protocol, an adjacent section of each of the tumors was stained with a mouse monoclonal antibody against factor-VIII-related antigen (clone F8/86; Dako), using the same protocol as above.
For optimization of R2–88 antibody dilution, serial R2–88 dilutions were performed to optimize signal-to-background ratio. The 1:6000 dilution had the highest immunohistochemical signal whereas the background remained low.
Formalin-Fixed, Paraffin-Embedded Tissues
As all tumors were primarily sent and processed for diagnostic purposes, it was usually not possible to standardize the fixation conditions and, therefore, to study in detail the effect of the fixation quality on the immunohistochemical signal, as fixation time and size of the specimen could vary from one case to another. In all tumors tested, the fixation time was, however, always on the order of 24 to 36 hours. In one case, the fresh tumor tissue was split into two parts, with one-half fixed in formalin for less than 24 hours and the other half fixed for 14 days. The fixed tissue was processed for conventional, 2- to 5-µm-thick paraffin (Paraplast) sections.
Effect of Pretreatment
Several different pretreatments were performed in a selected number of tumors to determine the optimal method for antigen retrieval in formalin-fixed tissue, according to the following protocols. Dewaxed and rehydrated tissue sections were 1) left untreated in TBS, 2) digested in 0.1% trypsin (Difco, Detroit, MI) in 50 mmol/L TBS, pH 8.0, with 10 mmol/L CaCl2 for 20 minutes at 37°C, 3) digested in 0.1% Pronase E (Sigma Chemical Co., St. Louis, MO) in 50 mmol/L TBS, pH 7.5, for 6 minutes at 37°C, 4) boiled in a total volume of 600 ml (3 x 200 ml) of 10 mmol/L citrate buffer, pH 6.0, in a microwave oven once for 8 minutes at 850 W and twice for 5 minutes at 410 W, followed by an additional period of 15 minutes in the hot buffer, and 5) immersed in 1.5 L of boiling 10 mmol/L citrate buffer, pH 6.0, in a pressure cooker that was then closed and slowly, over a period of 3 to 4 minutes, brought to 121°C. After a total time of 5 minutes, the pressure cooker was cooled under running tap water and opened, and the slides were transferred to H2O at room temperature for 5 minutes. After every pretreatment, slides were washed in TBS before the application of the primary antibody.
Signal Amplification
To enhance the immunohistochemical signal, the standard protocol, as described below, was followed by an additional amplification step: biotinylated tyramine was deposited onto the section through the activity of the bound peroxidase and subsequently served as a secondary target for another layer of avidin-biotin-peroxidase.23 We used both a commercial kit (Renaissance TSA-Indirect, NEN Life Science Products, Boston, MA), according to the manufacturer's directions, as well as an in-house system that had been developed according to Adams.24 Briefly, after the application of the avidin-biotin complex/horseradish peroxidase, slides were washed in TBS, incubated with 30 µmol/L biotinylated tyramine and 0.01% H2O2 in TBS, pH 8.0, for 15 minutes at room temperature, washed again in TBS, and incubated for 30 minutes with avidin-biotin complex/horseradish peroxidase. Finally, slides were developed with 3,3'-diaminobenzidine as above.
Standard Protocol for All Paraffin-Embedded Sections
Formalin-fixed, paraffin-embedded sections were dewaxed, rehydrated, and boiled in 10 mmol/L citrate buffer, pH 6.0, in a pressure cooker as described above. Sections were then (and after all subsequent steps) washed in TBS and incubated with the R2–88 polyclonal antibody against sst2A receptors overnight at room temperature. In formalin-fixed material, the antibody R2–88 was used at a dilution of 1:2000. All subsequent steps, including absorption of the antibody with the peptide antigen, were performed exactly as in the protocol for frozen tissue, and the same criteria were applied to distinguish between positive and negative tumors.
Preparation of Tumor Membranes and Immunoblotting
Frozen tumor tissue was homogenized in 1 ml of cold homogenization buffer (10 mmol/L Tris/HCl, 5 mmol/L EDTA, 3 mmol/L EGTA, 250 mmol/L sucrose, pH 7.6) containing protease inhibitors (1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml leupeptin, and 50 µg/ml bacitracin). After a low-speed centrifugation at 500 x g for 5 minutes, membranes were pelleted at 10,000 x g for 45 minutes. Membrane proteins from the sst2A-expressing rat growth hormone (GH)-producing pituitary cell line GH-R2 cells were prepared as previously described.25 After solubilization in sample buffer (62.5 mmol/L Tris/HCl, 2% sodium dodecyl sulfate, 10% 2-mercaptoethanol (v/v), 6 mol/L urea, and 20% glycerol, pH 6.8) at 60°C for 15 minutes, proteins were subjected to SDS-polyacrylamide gel electrophoresis on 7.5% polyacrylamide gels and transferred to polyvinylidene difluoride membranes.25 Receptor expression was determined by immunoblotting with a 1:10,000 dilution of R2–88.20
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Figure 1
demonstrates that the
antibody R2–88 recognizes a single protein in each tumor sample and
that reaction with this protein is blocked when the antiserum is
incubated with the peptide. In each case, this specifically stained
protein migrates as a diffuse band, consistent with glycosylation, and
is similar in size to the sst2A receptor expressed in a GH rat
pituitary tumor cell line (Figure 1)
as well as in Chinese hamster
ovary cells.20
The observation that the receptor protein
from different tumors migrates slightly differently indicates that
glycosylation is variable. However, as ligand binding was observed with
both of these tumors (see Table 2
), glycosylation does not have a major
effect on peptide recognition.
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Figure 2
shows the results obtained
with R2–88 using a standard immunohistochemical protocol for frozen
specimens. It represents a bronchial carcinoid tumor with two different
tumor parts: a central tumor region expressing only sst1 mRNA and
lateral regions expressing both sst1 and sst2 mRNAs, as illustrated
with in situ hybridization methods. 125I-labeled
Tyr3-octreotide binding is exclusively seen in the lateral
parts; the immunohistochemical staining for R2–88 is also detected in
these lateral parts, and only in these. In this positively
immunostained tumor, absorption with 100 nmol/L peptide antigen
eliminates completely the immunostaining, as a further proof of
specificity.
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shows that heat-induced epitope
retrieval methods, using either a pressure cooker or a microwave oven,
give a strong signal with R2–88; however, in the absence of such a
pretreatment as well as after Pronase or trypsin digestion of the
sections, R2–88 does not reveal sst2A-positive tumors. Tyramide
amplification gives a weak signal, which is, moreover, not completely
eliminated by absorption with 100 nmol/L peptide antigen. This suggests
that parts of the immunohistochemical staining with R2–88 in the
presence of tyramide is nonspecific, whereas the immunohistochemical
staining of the tumor (after boiling) appears highly specific. Figure 3
illustrates the various effects of
pretreatment, and Figure 4
shows the
effect of tyramide amplification on the R2–88 immunostaining of
sst2A-expressing neuroendocrine tumors. The incomplete specificity of
the tyramide amplification signal is clearly evident in this example.
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with two exocrine pancreatic tumors
that lack somatostatin receptor expression and that do not react with
R2–88 specifically. It is important to note that one case (Figure 5
,
right) shows a strong labeling of the tumor pancreatic ducts, which is
not abolished by preincubation of the antibody with the peptide
antigen. Therefore, some somatostatin receptor-negative tumors may
react nonspecifically with R2–88 serum. Thus, preabsorption with the
peptide antigen is a prerequisite for the specificity control of every
single tumor. As positive control, this same section (Figure 5)
shows
sst2-expressing normal pancreatic islets,26
which are
specifically stained with R2–88. As positive controls,
R2–88-immunoreactive sst2A receptors can also be identified in
germinal centers of lymphatic follicles27
located in the
surroundings of other somatostatin receptor-negative tumors (data not
shown).
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illustrates the specificity of
immunostaining in both frozen and paraffin-embedded sections. The left
column is an example of a gastrointestinal carcinoid, with abundant
sst2 mRNA as well as with a strong 125I-labeled
Tyr3-octreotide and 125I-labeled LTT-SS-28
binding; this tumor yields a positive immunohistochemical signal with
R2–88 in cryostat sections. Conversely, in the middle column, an
sst1-expressing leiomyosarcoma with abundant sst1 mRNA as well as with
strong 125I-labeled LTT-SS-28 binding, but lacking
125I-labeled Tyr3-octreotide binding as well as
sst2 mRNA, does not react with R2–88. Finally, the right column shows
an sst3-expressing insulinoma, with no 125I-labeled
Tyr3-octreotide binding and no sst2 mRNA but strong
125I-labeled LTT-SS-28 binding and abundant sst3 mRNA;
paraffin sections of this tumor are, as expected, not immunostained
with R2–88.
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documents that cryostat sections
or paraffin-embedded sections give similar results with R2–88 in an
example of an sst2-expressing glucagonoma. In addition, this figure
shows that the quality and intensity of the immunostaining with R2–88
is relatively insensitive to the fixation time with formalin, as a
14-day-long fixation is able to produce a positive immunostaining with
R2–88, comparable to a 12-hour fixation time.
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shows representative
immunostaining with R2–88 of a gastrinoma, using the above-described
standard protocol for paraffin-embedded sections pretreated with a
pressure cooker. It shows a precise immunohistochemical staining of the
tumor cells. The staining is completely abolished with antibody
absorption by 100 nmol/L peptide antigen. The sst2A receptors are seen
to be preferentially located on the cell membranes and expressed by the
great majority of the tumor cells. Note the specificity of the labeling
of tumor cells, as compared with adjacent ducts that are clearly
unstained. No marked difference in the immunostaining quality at the
cellular level is observed between boiling pretreatments using a
microwave oven or a pressure cooker. In those cases with a weak
immunostaining of the tumor cells, we have noticed that omitting the
counterstaining of the sections usually allows a clearer identification
of the membrane-bound receptor distribution, which is completely
abolished by peptide absorption, as expected.
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Tables 2 and 3
describe an extensive analysis of 47
tumors and compare the R2–88 immunohistochemistry with results of
other methods, including receptor binding and in situ
hybridization for sst mRNA. Table 2
shows that the antibody R2–88
detects immunohistochemically sst2A receptors expressed in cryostat
sections of human tumors. A positive immunohistochemical signal is
found in a variety of tumors, displaying a high-affinity binding for
both 125I-labeled LTT-SS-28 and 125I-labeled
Tyr3-octreotide. Some of these tumors had been shown by
in situ hybridization to contain sst2 mRNA as the only or
the predominant receptor subtype. Conversely, tumors expressing
somatostatin receptor subtypes different from sst2 by in
situ hybridization and having high-affinity binding only for the
universal ligand 125I-labeled LTT-SS-28 but not for
125I-labeled Tyr3-octreotide (Table 2)
do not
react with R2–88. Finally, none of the somatostatin receptor-negative
tumors, lacking both 125I-labeled LTT-SS-28 and
125I-labeled Tyr3-octreotide binding, react
with R2–88 (Table 2)
. Table 3
shows another series of human tumors,
where sst2A somatostatin receptors are detected by R2–88 in
formalin-fixed, paraffin-embedded sections. A positive
immunohistochemical signal is found in all tumors having both
125I-labeled LTT-SS-28 and 125I-labeled
Tyr3-octreotide binding and showing abundant sst2 mRNA.
Conversely, tumors expressing somatostatin receptor subtypes different
from sst2, ie, sst1 or sst3, and having high-affinity binding only for
the universal ligand 125I-labeled LTT-SS-28, but not for
125I-labeled Tyr3-octreotide (Table 3)
, do not
react with R2–88. None of a series of somatostatin receptor-negative
exocrine pancreatic tumors known to lack 125I-labeled
Tyr3-octreotide binding28
and to lack sst2
receptor mRNA17
react with R2–88 (Table 3)
. Furthermore,
an exact correlation is observed between the immunohistochemical
results in the paraffin-embedded sections and those in the cryostat
sections of a given tumor (Table 3)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These results show for the first time that the sst2A receptor protein is expressed in human tumors, as previous methods of analysis did not differentiate between the sst2A and sst2B isoforms; both bind octreotide and both splice variants are detected by in situ hybridization experiments. The results show that all tumors that were previously shown to express sst2 mRNA express the unspliced sst2A receptor protein. These results are in agreement with a recent RT-PCR study by Panetta and Patel11 showing that sst2A mRNA is frequently expressed in tumors. It is not yet clearly established whether sst2B protein is expressed in human tumors.15 As the two splice sst2 variants have been suggested to vary in their signaling properties and regulation,25,30,31 the expression of the sst2A form by human tumors is of functional significance.
To identify sst2A immunohistochemically, several technical requirements have to be considered. First, it should be stressed that the time of fixation in formalin does not seem to be crucial, as a comparable immunohistochemical signal is seen in the 12-hour and in the 14-day fixed tissue. However, it is essential to pretreat the formalin-fixed and paraffin-embedded sections for optimal antigen retrieval; without a specific pretreatment of the sections, no signal can be obtained. Only two pretreatments are efficient, namely, boiling treatments using a microwave oven or a pressure cooker; both give similarly good R2–88 immunostaining and the quality of the histopathological sections remains very good as well. Pronase and trypsin digestion treatment, however, do not allow detection of sst2A receptors with R2–88. Furthermore, the tyramide amplification method, which was successfully used in studies performed in intravitally perfused rat brain and pancreas,19,21 gave a relatively weak immunohistochemical signal and a high nonspecific signal in tumor and surroundings; in tumor tissues, therefore, the tyramide amplification was less satisfactory than boiling pretreatments for the sst2A receptor visualization.
The immunohistochemical evaluation of sst2A can be performed in cryostat sections as well as in paraffin-embedded material. This second option is of considerable interest for the pathologist as it gives for the first time the possibility to evaluate somatostatin receptors in the routinely processed archival paraffin-embedded material of any diagnostic pathology center.1 Although both methods are similar in terms of signal intensity, the paraffin-embedded sections have the advantage of a better histological quality and, therefore, better cellular resolution. With both methods, the membrane-bound localization of the sst2A receptors, as shown previously in rat brain with the same antibody,21 is particularly well identified. This membrane-bound localization of the receptors can be even more clearly recognized when the hematoxylin counterstaining is weak or omitted.
It is well established that the immunohistochemical use of most antibodies can give specific as well as occasional nonspecific staining. This applies also to the R2–88 antibody. In contrast to most conventional immunohistochemical procedures in diagnostic histopathology, it is therefore mandatory with R2–88 to perform in every single case a control experiment involving absorption of the antibody with the antigen peptide; only those tissues with complete abolition of the staining by saturating peptide can be considered to be sst2A receptor positive. If the peptide does not block the staining, the tissue is considered to be nonspecifically labeled, ie, sst2A receptor-negative, as seen in some somatostatin receptor-negative exocrine pancreatic cancers, for instance.
Although one can expect that the immunohistochemical detection of sst2A will be less sensitive than receptor binding with 125I-labeled Tyr3-octreotide, the sensitivity of R2–88 under the conditions developed here, ie, with adequate pretreatment, appears sufficiently good as all sst2 receptor-positive tumors of the present study were found to be sst2A immunoreactive and as normal human somatostatin target tissues, such as pancreatic islets26 or germinal centers of lymphatic follicles,27 were also unequivocally identified with R2–88.
This study shows for the first time that several methods, including receptor binding, in situ hybridization, and immunohistochemistry, can be combined and correlated to identify sst2A receptors in human tumors. The immunohistochemical identification of sst2A is therefore an important confirmation of the adequacy of all other previously performed in situ methods, ie, receptor autoradiography and in situ hybridization, used for many years for somatostatin receptor and somatostatin receptor subtype evaluation in various human tissues.1,9,16
The present investigation not only opens the gate for additional basic morphological investigations of sst2A receptors in human tumors and in normal human tissues, but it brings also, as an immediate consequence, a simple and rapid somatostatin receptor evaluation in the hand of the pathologist, with three major advantages. 1) This new method can analyze somatostatin receptors in paraffin-embedded tissues for the first time. 2) It requires only an immunopathological laboratory to perform the test and can be carried out without the complex and time-consuming receptor autoradiography or other techniques.1 3) The entire immunohistochemical procedure requires less than 24 hours. This new method is likely to be useful in the following situations: 1) differential diagnosis of selected tumors, ie, sst2-expressing endocrine versus sst2-negative exocrine pancreatic tumors,16,17 2) evaluation of the diagnostic potential of Octreoscan to visualize an individual tumor and its metastases, 3) evaluation of the potential clinical efficacy of octreotide and other stable sst2-preferring somatostatin analogues for the symptomatic therapy of gastroenteropancreatic and pituitary tumors, 4) evaluation of the potential for radiotherapy with radiolabeled octreotide analogues, and 5) evaluation of the prognosis of selected tumor types, in particular, neuroblastomas, which were shown previously to express somatostatin receptors of the sst2 type preferentially in those cases with favorable prognosis.32,33
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Footnotes |
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Accepted for publication April 14, 1998.
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